Highly efficient on-DNA amide couplings promoted by micelle forming surfactants for the synthesis of DNA encoded libraries

Abstract

DNA encoded libraries (DELs) represent powerful new technology for finding small molecule ligands for proteins and are increasingly being applied to hit finding in medicinal chemistry. Crucial to the synthesis of high quality DELs is the identification of chemical reactions for their assembly that proceed with very high conversion across a range of different substrates, under conditions compatible with DNA-tagged substrates. Many current chemistries used in DEL synthesis do not meet this requirement, resulting in libraries of low fidelity. Amide couplings are the most commonly used reaction in synthesis of screening libraries and also in DELs. The ability to carry out highly efficient, widely applicable amide couplings in DEL synthesis would therefore be highly desirable. We report a method for amide coupling using micelle forming surfactants, promoted by a modified linker, that is broadly applicable across a wide range of substrates. Most significantly, this works exceptionally well for coupling of DNA-conjugated carboxylic acids (N-to-C) with amines in solution, a procedure that is currently very inefficient. The optimisation of separate procedures for coupling of DNA-conjugated acids and amines by reagent screening and statistically driven optimisation is described. The generality of the method is illustrated by the application to a wide range of examples with unprecedented levels of conversion. The utility of the (N-to-C) coupling of DNA-conjugated acids in DEL synthesis is illustrated by the three cycle synthesis of a fully DNA-encoded compound by two cycles of coupling of an aminoester, with intermediate ester hydrolysis, followed by capping with an amine. This methodology will be of great utility in the synthesis of high fidelity DELs.

Fig. 1. Structures of headpieces: carboxy-PEG4-hexylamido-DNA 1, carboxy-C14-hexylamidoDNA 2 and amino-C11-hexylamidoDNA 3.

Fig. 2. Optimisation of temperature, surfactant strength (% TPGS) and base concentration by factorial experimental design, conditions: 2 (10 nmol), 2,6-lutidine, HATU (0.5 M), 30 μl total volume, 16 h; (a) cube plots showing modelled conversions; (b) % conversion responses for each parameter; (c) response surfaces showing the 2-dimensional relationship between conversion and temperature/base concentration. For full results see Table S2.

Fig. 3. Optimisation of temperature, surfactant strength (% TPGS) and base concentration by factorial experimental design, conditions: 2 (10 nmol), amine (0.5 M), 2,6-lutidine, DIC (0.5 M), 30 μl total volume, 3 h; (a) cube plots showing modelled conversions; (b) % conversion responses for each parameter; (c) response surfaces showing the 2-dimensional effect of temperature and surfactant concentration on conversion. Data shown are fitted using a least squares model (r² = 0.95, RMSE = 12). For full results see Table S6.

Fig. 4. Characterisation of reaction media by negatively stained transmission electron microscopy. (a) 2.5% aqueous TPGS-750-M; (b) 0.1 mM amino-C11-hexylamidoDNA 3 in water; (c) 0.1 mM amino-C11-hexylamidoDNA 3 in 3.5% aqueous TPGS-750-M.

Scheme 1. (a) Synthesis of representative encoded compound using 3 cycles of sequential amide couplings. Conditions: (i) glycine ethyl ester (0.5 M), 2,6-lutidine (1.5 M), DIC (0.5 M), HOAt (0.5 M), 4.5% TPGS, (30 μl), 45 °C, 3 h, then 0.25 M LiOH (0.25 M), 1 h, 63% overall yield; (ii) threonine methyl ester (0.5 M), 2,6-lutidine (1.5 M), DIC (0.5 M), HOAt (0.5 M), 4.5% TPGS, (30 μl), 45 °C, 3 h, then LiOH (0.25 M), 1 h, 27% overall yield; (iii) 3-bromopropargylamine (0.5 M), 2,6-lutidine (1.5 M), DIC (0.5 M), HOAt (0.5 M), 4.5% TPGS, (30 μl), 45 °C, 3 h, 75%. (b) Synthesis of representative encoded compound using 3 cycles of sequential amide couplings. Conditions: (i) ligation (primer and BB1 codon), then glycine ethyl ester (0.5 M), 2,6-lutidine (1.5 M), DIC (0.5 M), HOAt (0.5 M), 4.5% TPGS, (30 μl), 45 °C, 3 h, 94% yield for 2 steps; (ii) 0.25 M LiOH (0.25 M), 1 h, 100% yield; (iii) ligation (BB2 codon), then threonine methyl ester (0.5 M), 2,6-lutidine (1.5 M), DIC (0.5 M), HOAt (0.5 M), 4.5% TPGS, (30 μl), 45 °C, 3 h, then LiOH (0.25 M), 1 h, 51% yield for 3 steps; (iv) ligation (BB3 codon and closing primer sequence), then 4-fluoroaniline (0.5 M), 2,6-lutidine (1.5 M), DIC (0.5 M), HOAt (0.5 M), 4.5% TPGS, (30 μl), 45 °C, 3 h, 63% yield for 2 steps. Yields determined by Nanodrop™ spectrophotometry.